Introduction of Multi Myograph System 610M

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welcome
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Introduction of Multi Myograph System 610M

• Wu Liping
• 2004-6-4

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Background

• The technique was first developed for
  investigating small vessels with internal
  diameters down to around 100 µm by Mulcany
  Halpern , in 1976,1977
• Multi Myograph System 610M is suited to vessels
  with internal diameters of 100-400 µm
• At least 50 of the precapillary pressure drop
  occurs in the vessels with internal diameters
  than 100 µm ( proximal resistance vessels)

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General Principles

• Opening order
• powerlab myograph software
• Closing order
• software myograph powerlab
• Line connection
• one-to-one correspondingly

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General Principles

• Buttons function
• main options (zero/calibrate/temp
  erature, etc.)
• number changing
  (decrease/increase)
• F1 channel selecting
• F2 detailed operation
  

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General Principles

• 2 g weight

Calibration bridge

• Zero before experiments
• F 9.81 m N

tip
wire
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solutions
NAK KPSSNE 10 µ M
NAPSS PSSNE 10 µM
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dissection
Example

• The dissection of mesenteric small ateries from
  rats at autopsy
• mid-line laparotomy to exteriorize mesenteric bed
• Use scissors to remove 10cm of intestine together
  with its feeding vasculature, including part of
  the superior mesenteric artery. Proximal end of
  intestinal section should be 10cm from pylorus

• Place excised section in Petri dish containing
  PSS. Petri dish contains a 5 mm thick layer of
  Sylgaard to hold fixing pins.

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dissection

• Pin down the proximal end of the intestine on
  the left hand side of the dish, and pin out the
  remainder of the intestine in an anticlockwise
  direction
• Dissect out the test segment (normally the third
  branch) together with a piece of the proximal
  branch

• Using ocular dissection scissors, cut through
  the mesenteric membrane along either side of
  vessel, about 1-2mm from the vessel

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dissection

• Dissect away the vein (distinguish from the
  pattern of the branch points)
• Clean artery of connective tissue by holding the
  connective tissue with forceps and pulling gently
  away from the artery
• Cut distal end of vessel to be investigated and
  cut unwanted arcade 2mm from feeder artery, which
  are to be used as a handle to move the vessel
• Cut proximal end of feeder artery

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mounting

• Caution! The procedure involves attaching the
  mounting wires to jaws which are in turn mounted
  on the force transducer (sensitivity- 0.01 mN
  (1mg) upper limit- 1N (100g) )
• So ! Do not press the jaws too hard together a
  movement of 50mm after they have touched is
  sufficient to hold the wires clamped

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mounting
jaw
screw

• Attaching the first mounting wire
• Clamp the wire between two jaws
• Wrap the far end of the wire around under fixing
  screw clockwise and tighten the screw
• Fill myograph with PSS at room temperature

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mounting

• Mounting of the vessel
• Hold the handle segment of the excised arcade
  and try to mount the vessel onto the proximal end
  of wire.
• Pull the proximal end of excised arcade along
  wire until the vessel to be investigated is
  situated between the mounting jaws
• Screw jaws together to clamp the wire. Wrap the
  near end of wire around under near fixing screw
  again in a clockwise direction

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mounting

• Using forceps, gently rub vessel on the far side
  of the jaw and sever the vessel so that the
  portion of the excised arcade on the far side of
  the jaw can be pulled away
• Screw jaws apart, take a second wire and align it
  parallel with the vessel, passing into the far
  end of the lumen
• Screw jaws together. Ensure that the second wire
  moves under the first. Wrap the ends of wire like
  the former
• Screw jaws apart so that the wires should be
  levelled
• Connect oxygen line to chamber and start heating

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Normalization
T the myograph vessel supports had been moved
together (the mounting wires just touched)
B the vessel supports were moved slightly
apart, so that the record at B shows zero tension
1, 2, 3, and 4 the vessel was slowly
stretched, so that the records show the forces 1
min after each stretch. At 4, the vessel supports
were again moved together to release some of the
resting tension
N the vessel internal circumference was set to
IC1 (the internal circumference at which the
active force production of the vessel is maximal)
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Normalization
IC100 the vessels internal circumference when
it is fully relaxed and under a transmural
pressure of 100mmHg
IC1 0.9 IC100
Normalized lumen diameter is taken as I1 IC1/p
T wall tension, T pi(IC/2p)
Pi effective pressure, pi wall
tension/(IC/2p)
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Measurement of responses

• The response of a vessel to an agonist is
  normally presented in terms of the change in wall
  force above the resting force.
• F resting alpha(reading resting-reading
  baseline)
• F agonist alpha(reading agonist-reading
  baseline)
• Alpha is the force transducer calibration
  (mN/recorder division)

i.e. the force response ?FF agonist F resting
?F alpha(reading agonist-reading resting)
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removing Endothelium

• Purpose
• To remove the endothelium from an artery,
  trying to eliminate endothelial effects on the
  rest of the vessel
• To rule out the influence of the endothelium on a
  certain mechanism
• To confirm the role of the endothelium in the
  mechanism

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removing Endothelium

• Procedure
• First , fix the vessel and check the endothelium
  function
• Set the vessel to a small tension (0.5mN)
• Use a coarse pair of forceps to hold a human hair
  (clean, round, straight, thick), with a large
  microscope-magnification to look closely at the
  artery
• Enter the tip of hair into the vessel lumen, and
  work down through the hole inner surface of the
  vessel rubbing forwards and backwards

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removing Endothelium

• Procedure (cont.)
• Change to fresh solution in the chamber, and let
  the vessel equilibrate for 5-10 min
• Stretch the vessel to its normalized micrometer
  setting and let it relax for 5 min
• Check the endothelium-function again

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Checking endothelium-function

• Purpose
• To check whether the relaxing function of the
  endothelium is intact after mounting a vessel
• To check the success of an endothelium-removal
  procedure which you have applied to a vessel

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Checking endothelium-function

• Principle
• By stimulating a vessel with Acetylcholine
  (Ach.), the vessel will relax, following the
  release of EDRF (Endothelium Derived Relaxing
  Factor) from the endothelium.
• The procedure can be performed at any time after
  the heating and equilibration of the vessel.

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Checking endothelium-function

• Protocol 1 Quick check-single dose
• Stimulate 4 min with NA3PSS (10ml)
• Add 10 ml stock B to the chamber to give
  510(-6)M Ach
• Wait 2 min
• Wash out 4 times with PSS2.5

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Checking endothelium-function

• Protocol 2 Ach-concentration response curve
  (Ach-CRC)
• Stimulate 4 min with NA3PSS (10ml)
• Add 20 ml stock E to the chamber to give 10(-8)M
  Ach
• After 2 min, add 18 ml stock D to the chamber to
  give 10(-7)M Ach
• Repeat 3) with stock C and B to give 10(-6) and
  10(-5) M Ach
• After 2 min, add 90 ml stock A to the chamber,
  to give 10(-4)M Ach
• Wait 2 min
• Wash out 4 times with PSS2.5

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Checking endothelium-function

• Solutions
• NA3PSSPSS2.5noradrenaline 3 mM
• Stock AAcetylcholine (Ach) 10(-2)M (stock
  solution in freezer)
• Stock B Ach 5 10(-3)M . Made by mixing stock A
  and water 11
• Stock C Ach 5 10(-4)M . Made by diluting stock
  B 10-fold with water
• Stock D Ach 5 10(-5)M . Made by diluting stock
  C 10-fold with water
• Stock E Ach 5 10(-6)M . Made by diluting stock
  D 10-fold with water

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Thats all, thank you